Effects of Resveratrol on FOXO1 and FOXO3a Genes Expression in Adipose Tissue, Serum Insulin, Insulin Resistance and Serum SOD Activity in Type 2 Diabetic Rats

Induced oxidative stress in diabetes mellitus (DM) plays a critical role in insulin resistance. Fork head-related transcription factor (FOXO) proteins are important transcriptional factors involved in oxidative stress and insulin resistance. Resveratrol (RSV) is a polyphenol with hypoglycemic and antioxidant properties. The aims of the present study were to examine the effects of RSV on FOXO gene expression, serum superoxide dismutase (SOD) activity, insulin level, and insulin resistance in type 2 diabetic (T2DM) rats. Thirty male Wistar rats were used in this study. DM was induced in rats (n=24) using streptozotocin (STZ) and nicotinamide; then, they were divided into 4 groups of 6 rats each. Six untreated normal rats were used as normal control group; diabetic rats in groups 2 to 5 were treated with 0, 1, 5 and 10 mg /kg body weight of RSV, respectively for 30 days. At the end of the experimental period, the rats were sacriﬁced, their sera were separated, and adipose tissues were obtained and stored at −80 °C. Serum glucose and SOD activity levels were determined biochemically, and serum insulin level was determined by ELISA method. Gere expression in FOXO1 and FOXO3a in adipose tissue was evaluated using real‐time PCR. Results indicated that RSV significantly reduced blood glucose level, increased insulin level and improved insulin sensitivity. RSV resulted in an increased serum SOD activity and caused decreased FOXO1 and FOXO3a expression in adipose tissue of rats with T2DM. Therefore, by attenuation of FOXO expression in adipose tissue of T2DM rats, RSV showed a hypoglycemic potential and antioxidant properties, and consequently ameliorated insulin resistance.

iabetes mellitus (DM) is one of the most important metabolic disorders with impaired glucose, fat and protein metabolism, which contribute to a high morbidity and mortality worldwide (1). DM results in hyperglycemia due to defect in insulin secretion and/or insulin resistance (2). Hyperglycemia induces reactive oxygen species (ROS) production, leading to oxidative stress that D Submmited 02 September 2018; Accepted 13 November 2018; Published 31 December 2018 plays a critical role in DM-associated complications such as nephropathy, retinopathy and cardiovascular disease (1,3,4). Oxidative stress may lead to insulin resistance through several mechanisms. One of these mechanisms is the activation of forkhead box-related (FOXO) transcription factors (5). The FOXO family includes four transcription factors (FOXO1, FOXO3a, FOXO4 and FOXO6) that control the expression of genes involved in DNA repair, apoptosis, metabolism, oxidative stress, insulin resistance and longevity (6,7). The FOXO proteins play an important role in the metabolism, and are expressed in all tissues especially in adipose tissue, heart, brain, liver and skeletal muscle (5). These proteins play a significant role in insulin resistance and oxidative stress and their activity is regulated by silencing information regulators (sirtuins, SIRTs) (5). Deacetylation of this protein by SIRTs results in phosphorylation and inactivation of this transcription factor by SIRTs (8,9). Therefore, by affecting SIRTs (inactivation) DM results in acetylation and activation of FOXO and subsequent hyperglycemia, oxidative stress and insulin resistance. Thus, the activators of SIRTs or inhibitors of FOXO may improve hyperglycemia and oxidative stress and consequently insulin resistance (5). Today, herbal medicine has a particular role in disease treatment. Polyphenolic compounds such as resveratrol (RSV) has a beneficial effects on DM. RSV (3, 5, 4'trihydroxystilbene) is a polyphenol found in Polygonum cuspidatum, red grapes, red wine and berries (10). Many studies indicated that RSV has a hypoglycemic, anti-inflammatory and antioxidant effect (11,12). It has been established that RSV is a potent activator of SIRT1 and the activation of SIRT1 attenuates hyperglycemia (13). Additionally, due to the effect of RSV on SIRT1 activity, it can be concluded that RSV might have a beneficial effect on FOXO1, and FOXO3a expression in adipose tissue, and might consequently improve the oxidative stress and insulin resistance. The aim of the present study was to examine the effect of RSV on blood glucose level, FOXO1 and FOXO3a expression in adipose tissue, insulin level, insulin resistance and serum superoxide dismutase (SOD) activity in rats with T2DM.

Animals and study design
Thirty male Wistar rats (6-8 weeks old, To confirm the T2DM, 72 h after induction of diabetes, glucose level was assessed using a glucometer (Accuchek, Roche, Germany). The rats with blood glucose level higher than 150 mg/dl were considered as having T2DM (17). Seven days after T2DM induction, diabetic case groups, received 1, 5, and 10 mg/kg.bw/day doses of RSV, respectively. RSV was suspended in deionized water and administered using gavage at 10 am each day for 30 days. At the end of treatment period, the rats were anesthetized using ketamine: xylazine (100 mg/kg.bw : 5-10 mg/kg.bw, IP) and sacrificed (18). Blood sample was collected by cardiac puncture; serum was separated and stored at -20 °C.
Visceral adipose tissue was separated from each rat, cut into small pieces and was immediately frozen in liquid nitrogen and stored at -80 °C until analysis.

Determination of biochemical parameters in serum
The serum glucose level at day 37 was assessed by glucose oxidase method using a biochemical kit (Pars Azmun, Iran). Insulin was measured using rat insulin ELISA kit (Alpco, USA). Insulin resistance index (HOMA) was calculated by using "insulin (μU/ml) × glucose (mmol/l)/22.5" formula (19). The serum SOD activity was measured using a biochemical kit (Randox, England) and expressed as unit/ml.

RNA extraction and RT-PCR
The RNA extraction from adipose tissue was performed manually using TRIzol (Invitrogen, USA) according to manufacturer's protocol.
RevertAid first strand cDNA synthesis kit (Thermo scientific, USA) was used for cDNA synthesis using 1 µg of RNA. The SYBR Premix Ex Taq II (TaKaRa, Japan) was used to amplify the cDNA on a CFX96 Real-Time PCR detection system (BioRad, USA) and determine FOXO1 and   Normal control: healthy control group; Diabetic Control: diabetic untr. eated group; Diabetic+RSV 1 mg/kg: diabetic group treated with 1 mg/kg.bw/day of RSV; Diabetic+RSV 5 mg/kg: diabetic group treated with 5 mg/kg.bw/day of RSV; Diabetic+RSV 10 mg/kg: diabetic group treated with 10 mg/kg.bw/day of RSV. a: compared with normal control group; b: compared with diabetic control group; c: compared with diabetic rats that received RSV (1 mg/kg.bw/day); D0: before induction ofT2DM; D37: after completion of treatment period.*: P <0.05.

Correlation analysis
As indicated in Table 3 RSV is a polyphenolic compound, mostly known as an antioxidant and hypoglycemic agent (23).
Therefore, understanding the mechanism of RSV action provides beneficial information about its antioxidant and hypoglycemic effects.
In previous reports, we indicated that RSV has a powerful hypoglycemic effect and antioxidant properties (24,25